Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin 1A receptor.

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin 1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B 1 , an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin 1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin 1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.